Lectins are a class of proteins of non-immune origin that binds carbohydrates without modifying them. They are involved in many recognition events at molecular and cellular levels. Since lectins differ in the types of carbohydrate structures they can recognize they are used to detect and separate cells, bacteria, and viruses with different carbohydrate content. Lectins are also useful tools for investigating the structure, distribution and function of different carbohydrate chains on glycoproteins and glycolipids [Liljeblad et al., 2002; Rudiger et al., 2001; Yamashita et al., 1985].
The Aleuria aurantia lectin (AAL) from the fruit bodies of Aleuria aurantia mushroom has been extensively used in structural studies of oligosaccharides. AAL is specific for L-fucose and differs from other fucose-binding lectins by having a broad specificity towards fucosylated oligosaccharides [Debray et al., 1989; Fukumori et al., 1989; Kochibe et al., 1980; Nagata et al., 1991]. AAL can bind to oligosaccharides with fucose in α1-2, α1-3, α1-4 and α1-6 linkages, with the strongest affinity towards fucose in α1-6 linkage, but is relatively insensitive to structural differences in the oligosaccharide backbone [Debray et al., 1989; Wimmerova et al., 2003]. Since AAL is one of the few fucose-binding lectins with a preferential binding to α1-6 linked fucose it has been widely used in fractionation of glycoproteins with core-fucosylated complex-type N-glycans. Since changes in fucosylation is often associated with inflammatory conditions and oncogenic transformation AAL has also been used for fractionation and analysis of disease-associated glycosylation [Rydén et al., 1999; Rydén et al., 2002; Rydén et al., 2002]. Native AAL has been shown to agglutinate human erythrocytes of both A, B and 0 subtypes [Fukumori et al., 1989]
Recombinant AAL has been produced by expression in both E. coli and Pichia Pastoris, and subsequent purification. The recombinant forms of AAL have been shown to retain their agglutinating properties [Amano et al., 2003;].
AAL is a non-glycosylated protein that has a molecular weight of 72 kDa and is composed of two identical 312 amino acid subunits [Kochibe et al., 1980]. The lectin was recently crystallized and each monomer was shown to have a six fold β-propeller structure with five binding sites for L-fucose [Fujihashi et al., 2003; Wimmerova et al., 2003]. The slight structural differences at the five binding sites as well as the results from site specific mutagenesis studies indicated that the five possible binding sites for fucose differ in affinities towards fucose [Amano et al., 2003; Fujihashi et al., 2003; Wimmerova et al., 2003]. Site 2 and 4 seems to have the highest affinity towards fucose, site 1 to have medium affinity whereas site 3 and 5 seems to bind fucose with the weakest affinity [Fujihashi et al., 2003; Wimmerova et al., 2003].
Lectin-oligosaccharide interactions are generally characterized by a weak affinity (millimolar range) for monovalent binding. This low affinity is usually compensated by the fact that most lectins are multivalent. In contrast, several bacterial and fungal lectins have been shown to display unusually high affinity towards carbohydrate ligands compared to plant or animal lectins, with Kd-values in the micromolar range [Imberty et al., 2005; Kostlanova et al., 2005; Tateno et al., 2004]. A further understanding of the binding properties of these lectins will be important for designing high-affinity carbohydrate-binding proteins.
The multivalent nature of plant lectins is important for creating high avidity binding in nature. But the fact that most lectins show variation in binding affinity and binding specificity between different binding sites in the molecule presents problems, especially when plant lectins are used for diagnostic and preparative purposes.
Several diagnostic assays have been developed which measure pathological changes in carbohydrate composition using plant lectins as reagents [Hashimoto et al., 2004; Rydén et al., 1999; Rydén et al., 2002; Rydén et al., 2002]. However, since most target glycoproteins express multimers of the carbohydrate ligand and the lectins employed are multimeric in nature, linear relationships between expressed antigen and amount of bound lectin is seldom obtained. Thus these assays are usually only diagnostically relevant in a limited part of a concentration range.
There have been few previous attempts to produce monovalent carbohydrate-binding lectins. Procedures for preparing reduced valency Concanavalin A (a mannose and glucose-binding lectin) includes chemical modification such as succinylation and/or photoaffinity labelling (Fraser et al, 1976, Beppu et al 1976, Beppu et al 1975, Tanaka et al 1981, Gunther et al 1973,). Monovalent forms of Concanavalin A have also been prepared by proteolytic digestion (Wands et al 1976,). These methods were referred to in a previous patent application (WO9855869A1). Monovalent forms of the sialic acid-binding lectins Sambucus sieboldiana and Maackia amurensis as well as the galactose-binding lectin Anthocidaris crassispina and the Gal-NAc-binding lectin Wistaria floribunda have been prepared by disulfide-bridge reduction and subsequent protection with iodoacetamide (Kaku and Shibuya 1992, Kaku et al 1993, Ozeki 1991, Kurokawa 1976). These methods are not generally applicable to other lectins, and would not work to produce monovalent binding peptides from fucose-binding lectins such as Aleuria aurantia. No prior art of producing recombinant monovalent fucose-binding lectin peptides has been found. In a study of peptides containing GlcNAc-binding hevein domains Espinosa and co-workers [Espinosa et al, 2000] used a monovalent form of wheat-germ agglutinin—the isolated B-domain (WGA-B). They found that WGA-B retained its binding capacity towards chitotriose but that the binding affinity was too low to be considered useful for practical purposes (millimolar range).
Carbohydrate-binding peptides in prior art struggle with at least three problems arising from the multivalent nature of these peptides. Firstly, the problem of agglutination when carbohydrate-binding peptides bind more than one carbohydrate-expressing entity. This is a major drawback in cell surface e analysis of carbohydrates by flow cytometry, where concentrations of lectin have to be kept below agglutinating concentration, thereby significantly hampering sensitivity of the assay. Secondly, the problem of not achieving a linear relationship between carbohydrate expression and lectin-binding in more than just a limited part of a concentration range in an assay. Thirdly, the individual binding sites in multimeric lectins such as Aleuria aurantia differ in binding affinity and specificity towards carbohydrate ligands, which makes them unreliable for diagnostic purposes.